Modeling Tumor Angiogenesis with Zebrafish

The process of angiogenesis is essential for tumor progression and metastasis. New pathways have been identified to play a critical role in promoting and regulating blood vessel formation both in embryogenesis and in pathophysiological conditions. These pathways provide potential molecular targets for anti-angiogenic therapies to treat cancer and other vascular diseases. The critical cellular targets of these therapies are vascular endothelial cells (ECs) and supporting mural cells or pericytes (MCs) that are recruited from surrounding healthy tissue to form new vessels in the growing tumor. A challenging task has always been to visualize these biological processes in vivo as well as to screen for drugs affecting these pathological pathways. In this context, the zebrafish model represents an emerging vertebrate system to study the tumor angiogenesis process and to better understand the modification of tumor microenvironment by anti-angiogenesis therapy. Can a small tropical fish help to better understand the tumor angiogenesis process and identify new therapies for tumor angiogenesis ? In this chapter we illustrate how the zebrafish has emerged as a novel in vivo cancer model to study tumor-induced neovascularization and metastases. In the transparent zebrafish embryos, invasion and migration of tumor cells, their circulation in the vascular system, as well as the formation tumor-induced neovascularization can all be followed with high resolution in real time. Importantly, these zebrafish models allow to quantitate both metastatic behavior of transplanted tumor cells and tumor-cell induced neovascularization. The zebrafish model has the advantage of being a vertebrate equipped with easy and powerful genetic and imaging tools to investigate the mechanisms of tumor development and progression. In particular the transparency of embryos and lately also adult are transforming this model system in the leading in vivo model for cancer biology and tumor angiogenesis.


Introduction
Angiogenesis is process by which new blood vessels arise from endothelial cells in the existing vessels. In normal circumstances, the initiation, formation, maturation, remodeling and regression of endothelial cells in this process are strictly regulated. During tumor formation, the regulation of angiogenesis is disrupted and endothelial remodeling and regression are usually absent. Therefore, study on angiogenesis is of important relevance to cancer biology and therapeutic intervention (Carmeliet & Jain, 2000), especially in cancers where tumor growth depends on extensive vascularization (Folkman, 2002). A number of in vitro and in vivo models have been used for the study of angiogenesis. These include an endothelial cell line derived from human umbilical cord vein endothelial cells (HUVEC) (Jaffe et al., 1973) as well as a number of organ specific endothelial cell lines. With these cell lines, endothelial cell proliferation, differentiation and migration have been characterized. However, information about how endothelial cells interact with their neighboring cells is often lacking. In this regards, explant cultures (Brown et al., 1996;Jung et al., 2001) might be more representative of the complex interaction between endothelial and the supporting cells. Nevertheless, the issues of incomplete microenvironment, animal to animal variability and technical difficulties from relatively time-consuming and laborintensive tissue isolation and culture might limit the application of these models. In vivo models of angiogenesis have also been developed using chick embryo, rabbit and mouse (reviewed by Staton et al., 2009). They provide a more accurate physiological model of angiogenesis and when implanted with primary tumors or cancer cell lines, they can also provide important mechanistic insights to tumor angiogenesis. However, large-scale chemical screening with these models is difficult due to the cost and space needed for husbandry facilities. Zebrafish has emerged as a model organism for the study of genetics and human diseases. Compare with other vertebrate models, this small tropical fish offers distinctive advantages. Firstly, zebrafish embryos are externally fertilized and optically transparent, allowing direct visualization during embryonic development. Secondly, these embryos are amenable to reverse genetic manipulation including gene knock-down, over-expression or transgenesis by microinjection. Thirdly, the high fecundity of zebrafish enables adequate experimental duplicates and facilitates high through-put forward genetic screening. Mating a single pair of adult zebrafish can produce hundreds of eggs in one day. Fourthly, stable tissue-specific transgenic fish-lines are available, allowing direct visualization of various developmental processes. Lastly, husbandry and maintenance of zebrafish colonies are space and cost effective.
Early zebrafish embryonic vascular development begins at around 12 hour-post-fertilization (hpf) when hemangioblasts first exist along the lateral plate mesoderm. Later at around 24 hpf, the development of dorsal aorta (DA) and dorsal vein (DV), forming the first circulation loop. Subsequently, angiogenesis including the development of inter-segmental vessels (ISV) and sub-intestinal veins (SIV) occurs. Important growth factors and associated receptor tyrosine kinases as well as Notch signaling pathway regulating mammalian vascular development are conserved in zebrafish (Liang et al., 1998;Habeck et al., 2002;Goishi and Klagsbrun, 2004;Siekmann and Lawson ND, 2007). Here, we explore the potential of using zebrafish in vivo to model and more importantly to screen potential therapeutic agents targeting tumor angiogenesis.

Zebrafish embryonic angiogenesis
During zebrafish embryonic development, angiogenesis is characterized by the sprouting of inter-segmental vessels in the trunk between each somite initiated around 24 hpf as well as the development of sub-intestinal veins initiated around 48 hpf (Isogai et al., 2001;Lawson and Weinstein, 2002a). Although some argued the sprouting of ISV would represent type II vasculogenesis (Childs et al., 2002), these two processes are well accepted to represent early embryonic angiogenesis. Traditional assay to examine zebrafish angiogenesis includes alkaline-phosphatase (AP) staining of endothelial cells and whole-mount in situ hybridization of genes associated with vascular development such as fli1, flk1, flt4, efnb2a etc. Although in situ hybridization could provide more specific information such as artery or vein specification (Lawson and Weinstein, 2002a), these methods preclude direct and real-time visualization of the vasculature. Also, it takes days to complete staining protocols. These shortcomings have limited the application of zebrafish model until the recent advancement in zebrafish transgenesis and the availability of tissue-specific stable fluorescent reporter transgenic lines. With the use of fluorescent report transgenic zebrafish line such as Tg(fli1:egfp) (Lawson and Weinstein, 2002b) or Tg(flk1:egfp) (Jin et al., 2005), embryonic angiogenesis could be easily monitored real-time under fluorescent microscope. Figure 1 demonstrates the development of ISV and SIV at 48 and 72 hpf with Tg(flk1:egfp) and Tg(fli1:egfp).

Gene regulation of zebrafish angiogenesis
While angiogenesis is important for tumor growth and metastasis (Folkman, 2002), the precise mechanism and regulation of tumor angiogenesis remains unclear. Therefore, understanding angiogenesis during normal embryonic development might provide insight into how this process would be perturbed during tumor growth. Previous studies have demonstrated that genes that are involved in tumor angiogenesis such as galectin-1 (Thijssen et al., 2006), CXCR7 (Miao et al., 2007), angiomodulin (Hooper et al., 2009) and PDGFR-β/B-Raf (Murphy et al., 2010) may also play a role in embryonic angiogenesis. The zebrafish is unique in this respect because the circulatory system is dispensable during the first few days of embryonic development, enabling study of genes by specific knock-down that is otherwise lethal in the mammalian system.

Survivin and zebrafish angiogenesis
We have previously identified zebrafish survivin-1 (Ma et al., 2007a) as an important regulator of embryonic angiogenesis. Survivin exerts its effect through anti-apoptosis and interaction with VEGF receptor kinase pathway. Survivin is the smallest member of the inhibitor of apoptosis (IAP) gene family with a single Baculovirus IAP Repeat (BIR) domain and an extended -COOH terminal -helical coiled coil (Altieri, 2004). While it is not expressed in most normal adult tissues, survivin is highly expressed in solid and hematological malignancies, where it has been linked to tumor angiogenesis and represented a potential target for anti-cancer therapy (Graaf et al., 1998;Altieri, 2003). During human and murine embryonic development, survivin is ubiquitously expressed (Adida et al., 1998). However, homozygous knock-out of survivin in mouse ES cells results in disrupted microtubule formation and polyploidy as well as early embryonic fatality, precluding characterization of its functions during murine development (Uren et al., 2000) and therefore zebrafish embryo was considered an alternative embryonic model. In zebrafish embryos, survivin gene is duplicated into survivin-1 and survivin-2. During embryonic development, survivin-1 and survivin-2 are differentially expressed with distinctive functions in the vasculature and hematopoietic tissues (Ma et al., 2007a;Ma et al., 2009). Both survivin-1 and survivin-2 share a highly homologous functional BIR-domain and similar functions at cellular level. Therefore, the distinctive roles of survivin-1 and survivin-2 during embryonic development may be related to a large extent to their difference in spatial expression (Ma et al., 2009). In particular, survivin-1 predominantly expressed along the neural tube and axial vasculature at 26 hpf ( Figure 2). Knock-down of survivin-1 with anti-sense morpholino gives rise to defective angiogenesis as shown by defective spouting of ISV as well as SIV ( Figure 3). Vasculogenesis, demonstrated by the formation of axial vasculatures, was not affected. In vitro and tumorigenesis studies have shown that survivin mediates the angiogenic effects of VEGF (Tran et al., 1999;Mesri et al., 2001;Beierle et al., 2005). In zebrafish embryos, VEGF signaling is also important for angiogenesis. The schwentine mutant with defective VEGFR tyrosine kinase, flk1 (Habeck et al., 2002) has perturbed angiogenesis. In addition, phospholipase C- (plc-) mutant (y10) (Lawson et al., 2003) as well as knock-down morphant (Ma et al., 2007b) also exhibit specific defects in angiogenesis. VEGF induces ectopic angiogenesis and up-regulates survivin-1 mRNA expression (Figure 4a-c), suggesting that survivin-1 may mediate the angiogenic effect of VEGF. For instance, we only detect modest apoptotic TUNEL staining in the axial vasculature of survivin-1 morphant (Figure 4d, e) but not a direct causal link between increased apoptosis and the angiogenesis defect. VEGF might prevent apoptosis (Gupta et al., 1999) and VEGF inhibitors exert pro-apoptotic effect on endothelial cells (reviewed by Epstein, 2007). While apoptotic signal was readily detected along the neural tube of survivin-1 morphant (Figure 4d, e), survivin-1 might exert its antiapoptotic effect in a non-cell autonomous fashion downstream of VEGF, regulating the www.intechopen.com signaling cues for angioblasts to migrate from aorta to the dorsal aspect of the neural tube and to the inter-phase between notochord and the somites before ISV sprouting (Childs et al., 2002).

Zebrafish xenograft model of tumor angiogenesis
Recently, zebrafish xenograft models have been developed through xenotransplantation of human primary tumor cells or cancer cell lines into yolk sac of 48 hpf zebrafish embryos (Lee LM et al., 2005;Haldi et al., 2006;Topczewska et al., 2006;Nicoli et al., 2007;Marques et al., 2009). Without a functional immune system at this early embryonic stage, immunosuppression is not needed. The experimental procedures of transplanting fluorescent labeled human cancer cells into perivitelline space of 48 hpf zebrafish embryos was subsequently published (Nocoli and . In these models, cancer cells were shown to be engrafted into the yolk sac with proliferation and migration. More importantly, angiogenesis were induced in SIV with infiltration of blood vessels into the cancer mass. Combining with fluorescent reporter transgenic lines, these models serve as a promising platform to study the biology of tumor angiogenesis and its microenvironment including hypoxia (Lee SL et al., 2009) and LIM domain kinase 1 and 2 (Vlecken and Bagowski, 2009). www.intechopen.com

Screening potential therapeutic agents with zebrafish embryos 4.1 Large-scale chemical screening platform
Since angiogenesis is crucial for tumor growth and progression, anti-angiogenic agents have been investigated as potential anti-cancer therapies (Demetri et al., 2002;Cunnigham et al., 2004;Shepherd et al., 2005;Van et., al 2007;Hudes et al., 2007). Chemical screening based on in vivo tumor xenograft models are often limited by the relatively low throughput and long read-out time. In this respect, the zebrafish embryo is uniquely suitable for large-scale chemical screening because of the advantages aforementioned. In particular, using the Tg(flk1:egfp) or Tg(fli1:egfp) embryos, one could conduct large-scale in vivo screening against chemical libraries in a cost-effective way. To examine their effects on the initiation and regression of angiogenesis, embryos will be exposed to chemicals at different concentrations and developmental stages, either before angiogenesis (12 hpf), or after sprouting of ISV and development of SIV (48 hpf). Chemicals that specifically inhibit ISV and SIV formation after 12 hpf likely inhibit the initiation of angiogenesis and those that affect ISV and SIV after their formation at 48 hpf likely induce vascular regression ( Figure 5). Both anti-angiogenic mechanisms are considered important component in cancer therapy. This protocol may enable identification of potential anti-angiogenic compounds at high throughput and provide us with novel information about the link between embryonic and tumor angiogenesis. Figure 6 shows the use of Tg(flk1:egfp) embryos as a platform to demonstrate anti-angiogenic activity of VEGFR tyrosine kinase inhibitor and anti-cancer drugs (multi-kinase inhibitors) sorafenib and sunitinib.

Multiple chemicals can be tested at different concentration with hundreds of embryos easily available from mating
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Conclusion
Since angiogenesis is crucial for tumor growth and progression, it may present a potential target for cancer therapy. A number of anti-angiogenic agents targeting at the VEGF signaling pathway are being evaluated and large-scale chemical screening is needed to provide more candidates that can be tested in clinical trials. In this respect, the zebrafish embryos have emerged as a promising model that can shed important lights to the biology of physiological and tumor angiogenesis at whole organism level and allow cost-effective high throughput chemical screening. A number of new genetic modification technologies are now available that can specifically interrogate gene function related to angiogenesis. For instance, artificial endonucleases constructed by fusing non-specific nuclease domain with specific DNA binding domains (Ekker, 2008;Foley et al., 2009a;Foley et al., 2009b;Miller et al., 2011;Cermak et al., 2011;Sander et al., 2011) can now be used to target specific genes from zebrafish genome. An in vivo protein trap mutagenesis system (Clark et al., 2011) is www.intechopen.com also available that can simultaneously reveal spatio-temporal protein expression dynamics and assess gene function in zebrafish embryos. These new technologies greatly improve the efficiency of zebrafish genetic modifications and forward genetic screening, making zebrafish a more powerful model organism for angiogenesis.

References
Adida C, Crotty PL, Berrebi MD, Diebold J and Altieri DC. (1998). Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation. American Journal of Pathology, Vol.152, No.1, pp.43-49, ISSN 0002-9440 Altieri DC. (2003). Validating survivin as a cancer therapeutic target. Nature Reviews Cancer, Vol.3, No.1, pp.46-54, ISSN 1474-175X Altieri DC. (2004. Molecular circuits of apoptosis regulation and cell division control: Vasculogenesis is the process of new blood vessel formation during embryonic development of the cardiovascular system. This is followed by formation of a vascular tree and finally the cardiovascular system with the myriad of blood vessels that nourish all tissues and organs. Angiogenesis, on the other hand is the process by which new blood vessels take shape from existing blood vessels by "sprouting" of endothelial cells thus expanding the vascular tree. Both scenarios are based on activation, migration, proliferation and maturation of unique precursor cells. The study of blood vessel formation is an essential component of embryonic development, congenital malformations, degenerative diseases, inflammation and cancer and thus has widespread appeal to the biomedical field. Moreover, scientists are now harnessing this information for the purpose of building living blood vessel substitutes for replacement of diseased arteries and veins. This book highlights novel advances in the field of vasculogenesis and angiogenesis, including embryogenesis and development, regulation of progenitor cells, cancer and blood vessel regeneration. We consider this book a good initial source of information for graduate students, medical students and scientists interested in the intricacies of blood vessel formation, maturation, disease and replacement.